Abstract: Antibacterial resistance is a global public health crisis. Antibiotic-resistant Clostridium difficile is responsible for more than 29,000 deaths in US each year and the infection represents an urgent threat to public health worldwide. Of most concern is that the incidence of C. difficile infection (CDI) and disease severity is rapidly increasing in recent years due to the emergence of hypervirulent and antibiotic-resistant strains. CDI is caused by the exotoxins TcdA and TcdB secreted by C. difficile in the colon of the host. Current standard treatments with antibiotics are sub-optimal and accompanied by high rates of relapse due to the disruption of host colonization resistance. In contrast, animal and clinical studies have unambiguously demonstrated that colonization resistance and antitoxin antibodies are both highly protective against primary and recurrent CDI. In this SBIR Phase I project, we propose to develop a novel yeast immunotherapy. Our approach is to utilize an over-the-counter probiotic yeast Saccharomyces boulardii to deliver a potent toxin-binder for simultaneously targeting the two major virulence factors TcdA and TcdB for neutralization at the site of C. difficile infection. This single toxin-binding protein, designed as ABAB, consists of four non-overlapping neutralizing VHH antibodies, two against TcdA and two against TcdB. The hypothesis is that orally administered S. boulardii secreting ABAB in host intestines will relieve ongoing CDI and prevent recurrence. We have already constructed a prototype strain of S. boulardii for optimized secretion of ABAB (Sb-ABAB) under an antibiotic selection marker and demonstrated its therapeutic efficacy in a relevant animal disease model. In this project, we will generate chromosomal insertion of optimized ABAB expression cassette for stable secretion of ABAB in auxotrophic S. boulardii strain we generated and preclinically evaluate the therapeutic efficacy against both primary and recurrent CDI in mice and hamsters.